For evaluation of a target DNA sequence, a sample mixture is prepared containing one or more sets of sequencing polynucleotide fragments, each set containing fragments having lengths indicative of the positions of at least one base within the target DNA sequence. These sequencing fragment sets are each labeled with a different type of label (for example fluorescent labels). The sample mixture also includes a set of calibrant polynucleotide fragments having a plurality of known fragment lengths. The calibrant polynucleotide fragments are labeled with a spectroscopically-distinguishable calibrant label. The sample mixture is then electrophoretically separated to separate the polynucleotide fragments as a function of fragment length. Real-time detection is used to detect the label(s) on the set(s) of sequencing fragments and the calibrant label as they migrate in a common lane of the separation medium to produce a sequencing data trace and a calibrant data trace. The calibrant peaks are then used to define a set of coefficients for linearizing the sequencing data trace from each lane to a common corrected time scale in which the peaks from each lane are evenly spaced. The linearized sequencing data traces are then aligned by assigning base position numbers to each peak in the sequencing data traces, and these aligned traces are used for base calling.
Legal claims defining the scope of protection, as filed with the USPTO.
1. A method for evaluation of a target DNA sequence comprising the steps of: (a) preparing a first sample mixture comprising a first set of sequencing polynucleotide fragments having lengths indicative of the positions of a first type of base within the target DNA sequence, said first set of sequencing fragments being labeled with a first label, and a set of calibrant polynucleotide fragments having a plurality of known fragment lengths, said calibrant polynucleotide fragments being labeled with a calibrant label which is spectroscopically distinguishable from the first label; (b) electrophoretically separating the polynucleotide fragments in the first sample mixture as a function of fragment length in a separation medium; (c) detecting the first label and the calibrant label as they migrate in a common lane of the separation medium to produce a first sequencing data trace and a calibrant data trace; (d) generating a calibrant data set having a specified number of elements, each element comprising a base position number and a migration time for a peak in the calibrant data set; (e) fitting the calibrant data set to a polynomial having an order k to determine a first set of coefficients for linearization of a plot of migration time versus base position number, wherein k is an integer greater than 1, and the specified number of elements in the calibrant data set is at least equal to k 1; (f) resampling the first sequencing data trace at time intervals corresponding to a standard peak spacing defined by the polynomial and the determined coefficients to detect peaks in the sequencing data trace; and (g) creating a first sequencing data set comprising a number of elements equal to the number of peaks detected by resampling of the sequencing data trace, each element comprising at least a base position number for the peak which is be determined from the polynomial and the determined coefficients, wherein the first sequencing data set indicates the positions of bases of the first type in the target DNA sequence.
2. The method of claim 1 , wherein k is greater than or equal to 4.
3. The method of claim 1 , wherein the number of elements in the calibrant data set is greater than 6.
4. The method of claim 3 , wherein the number of elements in the calibrant data set is greater than or equal to 10.
5. The method of claim 4 , wherein k is greater than or equal to 4.
6. The method of claim 1 , further comprising the step of preparing one or more additional sample mixtures, wherein: (i) each additional sample mixture comprises a first additional set of sequencing polynucleotide fragments having lengths indicative of the positions of an additional type of base within the target DNA sequence, wherein the additional type of base may be the same as or different from the first type of base; (ii) the first additional set of sequencing fragments in each additional sampling mixture is labeled with a first additional label which may be the same as or different from the first label (iii) each additional sample mixture further comprises an additional set of calibrant polynucleotide fragments having a plurality of known fragments lengths, (iv) the additional set of calibrant polynucleotide fragments are labeled with an additional calibrant label which may be the same as or different from the first calibrant label and which is spectroscopically distinguishable from the first additional label in the same additional sample mixture; (v) each additional sample mixture is loaded onto a separate lane of the same separation medium as the first sample mixture and electrophoretically separated concurrently with the first sample mixture and detected to produce an first additional sequencing data trace and an additional calibrant data trace; (vi) an additional calibrant data set is generated from each additional calibrant data trace, and each additional calibrant data set is fitted to an additional polynomial having an order k to determine an additional set of coefficients for linearization of a plot of retention time versus base position number, wherein k is an integer greater than 1, and the specified number of elements in the additional calibrant data set is at least equal to k 1; (vii) calibrant data trace-specific scaling factors are determined for each lane of the separation medium, said scaling factors being selected such that multiplication of the scaling factor and the total run time of the associated calibrant data trace results in a constant value across all lanes of the separation medium; (viii) the first sequencing data trace is resampled at time intervals corresponding to a standard peak spacing defined by the polynomial and the determined coefficients multiplied by the associated scaling factor to detect peaks in the sequencing data trace; (ix) additional sequencing data traces are resampled at time intervals corresponding to a standard peak spacing defined by the associated additional polynomial and the determined additional coefficients multiplied by the associated scaling factor to detect peaks in the additional sequencing data traces; and (x) creating additional sequencing data sets each comprising a number of elements equal to the number of peaks detected by resampling of an additional sequencing data trace, each element comprising at least a base position number for the peak which is determined from the associated additional polynomial and the determined additional coefficients.
7. The method of claim 6 , wherein the first base an each additional base are different from one another, and wherein the first and additional sequencing fragment sets are different termination mixtures derived from the same source, further comprising the step of combining the first sequencing data set and the additional sequencing data sets to produce a combined sequencing data set that indicates the positions of bases of the first and additional types in the target DNA sequence.
8. The method of claim 1 , wherein the first sample mixture further comprises a second set of sequencing polynucleotide fragments having lengths indicative of the positions of a second type of base within the target DNA sequence, said second set of sequencing fragments being labeled with a second label which is spectroscopically distinguishable from the first label and the calibrant label, and wherein (i) a second sequencing data trace is obtained by detection of the second label, (ii) the second sequencing data trace is resampled at time intervals corresponding to a standard peak spacing defined by the polynomial and the determined coefficients to detect peaks in the second sequencing data trace; (iii) a second sequencing data set is created comprising a number of elements equal to the number of peaks detected by resampling of the second sequencing data trace, each element comprising at least a base position number for the peak which is be determined from the polynomial and the determined coefficients, wherein the second sequencing data set indicates the positions of bases of the second type in the target DNA sequence.
9. The method of claim 8 , wherein the first sequencing fragment set and the second sequencing fragment set are different termination mixtures derived from the same source, further comprising the step of combining the first sequencing data set and the second sequencing data sets to produce a combined sequencing data set that indicates the positions of bases of the first and second types in the target DNA sequence.
10. The method of claim 9 , further comprising the steps of preparing an additional sample mixture comprising first and second additional sets of sequencing polynucleotide fragments having lengths indicative of the positions of a first and second additional types of base within the target DNA sequence and an additional set of calibrant polynucleotide fragments having a plurality of known fragment lengths, wherein the first and second additional bases are different from each other and from the first and second types of bases whereby all four base types are represented and wherein the first and second additional sets of sequencing fragments and the additional set of calibrant fragments are each label with a first and second additional labels and an additional calibrant label, said labels being spectroscopically distinguishable from each other; loading the additional sample mixture onto a lane of the same separation medium as the first sample mixture and separating the additional sample mixture concurrently with the first sample mixture; obtaining first and second additional sequencing data traces and an additional calibrant data trace by detecting the first and second additional labels and the additional calibrant label as the fragments migrate through the separation medium; generating an additional calibrant data set having a specified number of elements, each element comprising a base position number and a migration time for a peak in the calibrant data set; fitting the additional calibrant data set to an additional polynomial having the order k to determine an additional set of coefficients for linearization of a plot of retention time versus base position number, wherein the specified number of elements in the additional calibrant data set is the same as in the first calibrant data set; determining a scaling factor for the additional polynomial, said scaling factor being selected to equalize the total run time of the first calibrant data trace and the additional calibrant data trace; resampling the first and second additional sequencing data traces at time intervals corresponding to a standard peak spacing defined by the additional polynomial and the determined coefficients, multiplied by the scaling factor, to detect peaks in the first and second additional sequencing data traces; creating first and second additional sequencing data sets, each comprising a number of elements equal to the number of peaks detected by resampling of the first or second additional sequencing data trace, respectively, each element comprising at least a base position number for the peak which is be determined from the polynomial and the determined coefficients, multiplied by the scaling factor, wherein the first additional sequencing data set indicates the positions of bases of the first additional type in the target DNA sequence and the second additional sequencing data set indicates the positions of bases of the second additional type in the target DNA sequence; combining the first and second additional data sets with the combined data set to produce a complete combined data set that indicates the positions of all four types of bases in the target DNA sequence.
11. A data analysis unit for use in analysis of DNA sequence data obtained using an electrophoresis unit, said DNA sequence data including at least one sequencing data trace and one calibrant data trace per lane, said data analysis unit comprising, (a) an input means for receiving the DNA sequence data; (b) a processor for processing the DNA sequence data to produce a linearized and aligned data set; and (c) an output connector for communicating the linearized and aligned data set to a user; wherein the processor is programmed to perform the steps of: selecting peaks from each calibrant data trace for fitting; fitting the selected peaks to a polynomial function to determine a coefficient set for each calibrant data trace effective to linearize a plot of peak number versus migration time; determining a scaling factor for each calibrant data trace, said scaling factors being selected such that they normalize the total run time of the calibrant data traces to a common value; applying the polynomial with the determined coefficients and the scaling factor for each calibrant to the sequencing data trace(s) from the same lane to generate a peak set N, T iN in a corrected time domain; and when the DNA sequence data includes two or more related sequencing data traces, combining the peak sets generated for the related data traces into a combined peak set for base calling.
12. An integrated apparatus for sequencing of nucleic acids comprising: (a) an electrophoresis unit; and (b) a data analysis unit in accordance with claim 11 .
13. The apparatus of claim 12 , further comprising at least one output device.
14. The apparatus of claim 13 , wherein the output device is a display.
15. The apparatus of claim 13 , wherein the output device is a printer.
16. The apparatus according to claim 12 , wherein the electrophoresis unit comprises a detector system effective to detect three different spectroscopically distinguishable labels per lane.
17. The apparatus of claim 16 , wherein the detector system comprises a detector module for detection of light transmitted by or emitted from a sample, wherein the light, depending on the nature of the sample, may include light of up to three spectroscopically-distinguishable wavelengths, and wherein the detector module comprises: three optical bandpass filters, one for each spectroscopically-distinguishable wavelength, each of said bandpass filters transmitting light of one of the spectroscopically-distinguishable wavelengths and reflecting light of other spectroscopically-distinguishable wavelengths; and three photodetectors, wherein the bandpass filters are disposed in an arrangement such that light which is not transmitted by a bandpass filter is reflected to impinge on a successive bandpass filter in the arrangement, and wherein each of the photodetectors is disposed to detect light which has been transmitted by a different one of the bandpass filters.
18. The apparatus according to claim 17 , wherein the three bandpass filters transmit light of 660-680 nm, 685-715 nm and 730-770 nm, respectively.
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July 27, 2000
May 28, 2002
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